With more than 1.2 million cases diagnosed each year, breast cancer is the most common malignancy in women resulting in approximately 500,000 deaths per year worldwide with 90% of these deaths due to metastasis. While advances in gene expression profiling of primary tumor cells have recently led to new tools with some prognostic power for recurrence, there are few predictors of actual metastatic risk. After more than 10 year of investigation into the tumor microenvironment (epitomized by the Tumor Microenvironment Network program at the National Cancer Institute) it is becoming increasingly clear that, in addition to driven mutations, the tumor microenvironment determines tumor metastatic phenotype.
This research has led to a new understanding of the impact of the tumor microenvironment heterogeneity upon proliferation, and more importantly, dissemination. In previous studies, certain of the inventors examined the role that tumor micro-environmental parameters (hypoxia, stromal and immune cells, extracellular matrix, stemness) have on breast cancer cell dissemination and dormancy in vivo, and at single cell resolution, using novel multiphoton imaging tools and intravital imaging techniques. This research highlights how high-resolution imaging can identify, localize, and quantify heterogeneity in the tumor microenvironment, in vivo, and reveal cell-cell interactions and mechanisms that cannot be observed using fixed tissue, i.e., tissue samples which have been cross-linked in paraformaldehyde (PFA) and then, stained for selected antibodies to allow for imaging by microscopy. A full understanding of this heterogeneity, both temporally and spatially, in the primary and secondary sites; how it supports tumor cell dissemination, dormancy and eventual further metastatic growth; and how it responds to therapeutic interventions, is crucial since it can reveal commonalities and differences that could lead to unique treatment approaches and therapies.
To accomplish this, experiments designed to identify, locate, and characterize the function of the cells contributing to this microenvironment need to be performed at widely varying temporal and spatial scales (from minutes to weeks and from sub-cellular to tissue wide) and at vastly different stages (initiation on to metastasis), in order to give a complete understanding of tumor progression. Unfortunately, this understanding has been delayed by significant limitations of approaches that are currently employed.
Conventional tools like 2D in vitro assays do not adequately reflect the topography encountered by cells in vivo. Even 3D in vitro assays which remove this restriction on topography, still lack the diversity and heterogeneity of environments present in the living organism (e.g. multiple host cell interactions, physiological extracellular matrix, connection to lymphatic and vascular circuits, etc.). Thus in vivo methods are essential; however quantitation of cell subsets in in vivo tissues is typically accomplished either by histology or FACS (Fluorescence Activated Cell Sorter) analysis. These methods are also limited as they can only be used in single time point, end-stage experiments, with FACS additionally disrupting the tissue spatial arrangement.
Identification of cell types can alternatively be accomplished by using genetically modified mouse models. Unfortunately, these models take months to years to develop and are not applicable to human tissues. Further, those experiments that are performed in living animals are typically limited to systemic applications of drugs, functional blocking antibodies, or inducible genetic alterations that are un-localized and create many off-target effects that can confound experimental results. Furthermore, analyses of these approaches again relies on end-stage assays of fixed tissues followed by histology or FACS.
What is needed is the ability to visualize, identify and manipulate specific cell types and their dynamics while they are resident in the living tissue at both the primary and various secondary sites.
Existing intravital windows facilitate repeated imaging in vivo but generally inhibit introduction of drugs or other fluids to adjacent target tissue after window installation. US published application 2014/0308207 discloses an on-chip microfluidic device (OMD) providing microscopic observation and creating gradients in the underlying tissue. However, the OMD employs constant one-way flow, generated by syringe pumps, to diffuse chemoattractant solution into tissue, and requires tubing and a housing structure for importing the solution from the external environment. Further, the OMD is constrained to release its chemoattractant solution through all of its outlets simultaneously and at one time.
A more compact, efficient, remotely controllable and versatile microfluidic intravital window is desirable.